1. Field of Invention
This invention relates to the allosteric regulation of protein function through stabilization of one or more different quaternary assemblies, herein defined as morpheein forms. The different morpheein forms of a given protein have different surface characteristics that can be targeted for the development of a broad spectrum of bioactive agents. As an example, this invention relates to the biosynthesis of tetrapyrroles, and more particularly to a mechanism for inhibiting activation of porphobilinogen synthase.
2. Description of Related Art
It is generally accepted that the three dimensional structure of a protein is determined by the sequence of that protein and that there is one structure for each protein [1]. Exceptions to this rule include prions, a term that remains controversial, but is taken to refer to proteins that can change from their biologically active globular shape to a fibrillar shape which can form an aggregate that can grow indefinitely into disease causing amyloid plaques (e.g. scrapie). The formation of the amyloid protein structure from the globular prion protein structure is believed to be irreversible. This invention does not deal with prion proteins, nor with any other irreversible transformation in protein shape, such as denaturation. A second example of a situation where one protein is known to exist in two forms, more particularly in two quaternary assemblies, occurs as the quasiequivalence of some virus capsid proteins [2,3]. The quasiequivalent virus capsid proteins are the stable components of the geodesic dome that encapsulates the virus; these stable structures exist as parts of a larger assembly and are fundamentally different from the morpheein structures introduced herein.
Allosteric effects on ligand binding and/or catalytic activity are changes in said binding or activity which are caused by binding of another molecule (the allosteric effector molecule) to a site on the protein that is different from the ligand binding site or active site. The behavior of many proteins is known to be subject to allosteric regulation. Many allosteric proteins are known to exist as homomeric multimers (oligomers made up of subunits where the sequence of each of the subunits is identical to the others). There are two well-accepted models for allosteric regulation, the Monod Wyman Changeux model and the Koshland model [4,5]. Both of these models explicitly presume that the allosteric “ON” state and the allosteric “OFF” state have a constant oligomeric multiplicity as illustrated schematically in FIG. 1. The current invention is about a third model for allosteric regulation of protein function, the morpheein model, where the oligomeric multiplicity of the “ON” state and the “OFF” state of a homo-oligomeric protein are different.
Tetrapyrrole biosynthesis is an essential pathway in animals, plants, and microbes, including bacteria, archae, fungi, and protists. The first common intermediate is 5-aminolevulinic acid (ALA). The enzymatic reactions from ALA to uroporphyrinogen are common to tetrapyrrole biosynthesis in all organisms [6,7].
The enzyme porphobilinogen synthase (PBGS, EC 4.2.1.24), also known as 5-aminolevulinic acid dehydratase (ALAD), is an ancient and highly conserved protein that catalyzes the first common step in the biosynthesis of tetrapyrroles including heme, chlorophyll, vitamin B12, and cofactor F430 [8,9]. PBGS catalyzes the condensation of two 5-aminolevulinic acid molecules to form the tetrapyrrole precursor porphobilinogen.
PBGS was previously understood to be a homooctameric metalloenzyme, which utilizes a variety of divalent and monovalent cations at catalytic and allosteric sites. The first 18 deposited X-ray crystal structures showed an octameric assembly [10], as illustrated for human PBGS in FIG. 2. Mammalian and yeast enzymes typically require Zn(II), some prokaryotic enzymes require either Mg(II) or Zn(II) or both for maximal activity, and plant enzymes seem to require only Mg(II) for enzymatic activity. A small number of organisms have PBGS enzymes that require neither Zn(II) nor Mg(II). The difference in the use of metal ions is caused by a variation of residues in the primary structures in at least two metal-binding regions [11]. The structure of E. coli PBGS is illustrated in FIGS. 3A-C and serves to illustrate the common metal binding variations in PBGS structures. Each E. coli PBGS monomer contains two metal ions, neither of which is phylogenetically conserved. The active site contains a zinc ion that is essential to E. coli PBGS activity but whose three cysteine ligands are not present in many PBGS. This zinc functions in the binding and reactivity of the second substrate molecule [12]. Details of the zinc site are illustrated in FIG. 3B. In addition, there is an allosteric magnesium that is seen bound at the interface of each alpha, beta-barrel with the N-terminal arm of a neighboring subunit; structural details are in FIG. 3C. The sequence determinants for binding the allosteric magnesium are not present in all PBGS. The PBGS have been categorized into four groups based on whether or not they have the catalytic zinc binding site and whether or not they have the allosteric magnesium binding site, as illustrated in FIG. 4.
FIG. 4 is a schematic for classifying the PBGS into four groups on the basis of whether or not they use an active site zinc and whether or not they use an allosteric magnesium [11]. The first matrix (far left) is divided into two classes: (a) active site zinc on the left (shaded), and (b) no active site zinc on the right (unshaded). The second matrix is divided into two classes: (a) no allosteric magnesium on top (white), and (b) allosteric magnesium on the bottom (squares). Combining the two matrixes provides a matrix (far right) consisting of four quadrants, wherein the northwest quadrant (QNW) represents +Zn/−Mg, the northeast quadrant (QNE) represents −Zn/−Mg, the southwest quadrant (QSW) represents +Zn/+Mg, and the southeast quadrant (QSE) represents −Zn/+Mg. The terms QNR, QNR, QSW, and QSE are used throughout this document to refer to the quadrants of FIG. 4.
The inventor has previously quantified [11] the following distribution of known sequences into the four quadrants: QNW=9; QNE=2; QSW=55 and QSE=63. Thus, approximately one-half of the currently available sequences encode an active site zinc requirement and one-half do not (i.e., QNW+QSW˜QNE+QSE). In contrast to the active site metal pattern distribution, more than 90% of the PBGS sequences contain the determinants for allosteric magnesium binding (i.e., QSW+QSE>>QNW+QNE).
It has been found that the specific activity of PBGS from some sources is dependent on protein concentration, as illustrated in FIG. 5. For example, a protein concentration dependence for the specific activity has been seen for B. japonicum, P. aeruginosa, R. capsulatus and pea PBGS, but has not been documented for PBGS from E. coli, yeast, or from mammalian sources [13-15]. Prior interpretation of this phenomenon was a simple dissociation reaction of a maximally active octamer to lesser active or inactive tetramers and/or dimers (FIG. 5). Prior interpretation did not include alternative morpheein forms of PBGS.
It is known to inhibit PBGS by removing metals from an active site or from an allosteric site, e.g., by treating it with ethylenediaminetetraacetic acid (EDTA), or 1,10-phenanthroline [16,17].
Today, many consumers are demanding that personal health care products such as wet wipes, diapers, etc. have the ability to not only provide their intended function, but to cure or prevent a disease or a damage caused by contacting bacteria, archaea, and/or eucarya, for example, while not harming the consumer's health. To meet this demand, antimicrobial agents have been incorporated into a wide range of consumer products, such as wet wipes, to combat both transient and resident bacteria on skin. Antimicrobial-containing products are currently marketed in many forms such as lotions, deodorant soaps, hard surface cleaners, wet wipes, and surgical disinfectants.
Biofilms can be a problem for certain surfaces. Biofilms may be found on essentially any environmental surface in which sufficient moisture is present. Their development is most rapid in flowing systems where adequate nutrients are available. Biofilms are composed of populations or communities of microorganisms adhering to environmental surfaces and are complex aggregate of cells and polysaccharide. These microorganisms are usually encased in an extracellular polysaccharide that they synthesize. The biofilm, for example can be formed from mixed culture of Pseudomonas aeruginosa, P. fluorescens and Kiebsiella pneumoniae. Biofilms may form on solid substrates in contact with moisture, on soft tissue surfaces in living organisms and at liquid air interfaces. Typical locations for biofilm production include rock and other substrate surfaces in marine or freshwater environments. Biofilms are also commonly associated with living organisms, both plant and animal. Tissue surfaces such as teeth and intestinal mucosa which are constantly bathed in a rich aqueous medium rapidly develop a complex aggregation of microorganisms enveloped in an extracellular polysaccharide they themselves produce. The ability of oral bacteria to store iodophilic polysaccharides or glycogen-like molecules inside their cells is associated with dental caries since these storage compounds may extend the time during which lactic acid formation may occur. It is this prolonged exposure to lactic acid which results in decalcification of tooth enamel.
People have made use of microbial biofilms, primarily in the area of habitat remediation. Water treatment plants, waste water treatment plants and septic systems associated with private homes remove pathogens and reduce the amount of organic matter in the water or waste water through interaction with biofilms. On the other hand biofilms can be a serious threat to health especially in patients in whom artificial substrates have been introduced. Also, biofilms are a threat to bottoms of ship wherein barnacles can grow and corrode the surface or on the external or the internal surfaces of pipes such as oil pumps or dehumidifiers.
As more has been learned about the differences in sequence and structure for various proteins/enzymes, it has become possible to target an essential pathway that is universally present in animals, plants, bacteria, and fungi. Such is the case for targeting the tetrapyrrole biosynthetic pathway through the inhibition of PBGS as the foundation for antimicrobials or herbicides. The phylogenetic variation in metal binding sites among the PBGS of various organisms provides sufficient structural differences for development of an inhibitory agent that will not be inhibitory toward human PBGS. In the case of PBGS, there are significant differences between organisms in the inherent ability of the PBGS to equilibrate between morpheein forms and in the amino acid sequence of the morpheein surfaces. In the case of the more general inhibition of protein function through the selective stabilization of one morpheein form, it may be the case that the target is a pathway that is not present in humans or it may be the case that the target simply has sufficient phylogenetic variation outside the active site that the surfaces of the morpheein forms are very different. For instance, sequence conservation in proteins is highest in the region of shared function, as in an enzyme active site. Sequence conservation is not high in regions that are not involved in shared function. Protein surfaces are the most susceptible to evolutionary changes and the least likely to be conserved between an organism (e.g. human) and it pathogen.
Accordingly, the inventor has developed bioactive compositions having universal applications, and methods for identifying such compositions, as well as methods of identifying proteins which are homo-oligomeric and allosteric in nature, and which will serve as targets for bioactive compounds that will inhibit or activate these homo-oligomeric proteins through perturbation of an equilibrium of quaternary assemblies. It is further desired to provide an agent capable of disturbing an equilibrium of units of multimeric proteins, e.g., an inhibitor capable of inhibiting tetrapyrrole biosynthesis in plants and/or bacteria through the stabilization of a lesser active quaternary assembly of porphobilinogen synthase. It is further desired to accomplish such inhibition via a mechanism that is also applicable to humans and animals, thereby creating a novel, highly specific, approach to bacteriostatic, antibiotic, or herbicide activity. As many essential proteins are homo-oligomeric and allosteric in nature, it is desired to provide bioactive compounds that will inhibit or activate these homo-oligomeric proteins through perturbation of an equilibrium of quaternary assemblies. The inventor has identified a compound, termed morphlock-1 (see FIGS. 32a and 32b) which was found to dramatically shift the equilibrium of quaternary assemblies of multimeric proteins.
All references cited herein are incorporated herein by reference in their entireties.